The monomeric archaeal primase from Nanoarchaeum equitans harbours the features of heterodimeric archaeoeukaryotic primases and primes sequence-specifically

Abstract

The marine thermophilic archaeon Nanoarchaeum equitans possesses a monomeric primase encompassing the conserved domains of the small catalytic and the large regulatory subunits of archaeoeukaryotic heterodimeric primases in one protein chain. The recombinant protein primes on templates containing a triplet with a central thymidine, thus displaying a pronounced sequence specificity typically observed with bacterial type primases only. The N. equitans primase (NEQ395) is a highly active primase enzyme synthesizing short RNA primers. Termination occurs preferentially at about nine nucleotides, as determined by HPLC analysis and confirmed with mass spectrometry. Possibly, the compact monomeric primase NEQ395 represents the minimal archaeoeukaryotic primase and could serve as a functional and structural model of the heterodimeric archaeoeukaryotic primases, whose study is hindered by engagement in protein assemblies and rather low activity.

Definition of the binding specificity of the T7 bacteriophage primase by analysis of a protein binding microarray using a thermodynamic model

“…Thus, a motif is present on average every 2 2.5 (∼6) nucleotides. In a recent characterisation of the priming specificity of the archaeoeukaryotic primase from Nanoarchaeum equitans , the priming specificity was found to be approximately 3 bits and the binding specificity can be expected to be even lower ( 31 ) underscoring the difficulties in determining the primase binding specificity in contrast to the priming specificity.…”

Initial Primer Synthesis of a DNA Primase Monitored by Real-Time NMR Spectroscopy

“…For example, PriS and PriL in Saccharolobus solfataricus form a complex with a third small subunit, PriX. 8,9 In contrast, the plasmid pRN1 primase in Sulfolobus islandicus and the primase from Nanoarchaeum equitans 10 are monomeric primases encompassing the domains homologous to PriS and PriL. PriX in Saccharolobus solfataricus and the C-terminal domain of pRN1 primase in Sulfolobus islandicus as well as the C-terminal domain of the Nanoarchaeum equitans primase fold into a helix-bundle domain (HBD), which is a structural orthologue of the C-terminal domain of the eukaryotic PriL.…”

“…Another difference of archaea compared to eukaryotes is that DNA primases not only exist as a heterodimer but also can occasionally be found as a trimer or as a single protein containing two separate domains. For example, PriS and PriL in Saccharolobus solfataricus form a complex with a third small subunit, PriX. , In contrast, the plasmid pRN1 primase in Sulfolobus islandicus and the primase from Nanoarchaeum equitans are monomeric primases encompassing the domains homologous to PriS and PriL. PriX in Saccharolobus solfataricus and the C-terminal domain of pRN1 primase in Sulfolobus islandicus as well as the C-terminal domain of the Nanoarchaeum equitans primase fold into a helix-bundle domain (HBD), which is a structural orthologue of the C-terminal domain of the eukaryotic PriL. , The HBD of all these proteins have been suggested to be the binding site for the initiating nucleotides during primer synthesis, and deletion of these domains abrogates primase initiation but not elongation. ,, However, the exact mechanism by which DNA primases transfer these initiating nucleotides from the HBD to the active site, which is located in PriS, is still unknown.…”